Specific mutated proteins in influenza A may be important diagnostic markers related to the virulence of pandemic strains. Analysis of the PB1-F2 sequence identified a single mutation (N66S) that was conserved for both the Hong Kong 1997 H5N1 highly pathogenic influenza as well as the 1918 pandemic strain. Additional studies have demonstrated that the NA stalk region of N1 influenza A viruses also affects virulence. The ability to rapidly screen for these, and other yet-to-be- discovered virulence factors, would significantly improve our ability to identify emerging strains with risk of high mortality, and allw public health professionals to effectively respond to prevent widespread infection and/or death. Unfortunately, a rapid, multiplexed method to screen for virulence factors does not exist. Recent research in our laboratories has demonstrated that a nanofabrication technique based on vapor deposition produces Ag nanorod arrays that exhibit extremely high electromagnetic field enhancements when they are used as surface-enhanced Raman spectroscopy (SERS) substrates. Our initial results suggest that these novel SERS substrates can be used as diagnostic tools to rapidly and sensitively detect the Raman spectra of RNA and DNA viruses. SERS spectra effectively act as molecular fingerprints that provide specific and quantitative information about the target. Using this method, we have shown that differentiation is possible between virus strains, serotypes, genotypes and oligonucleotides based solely on their intrinsic SERS spectra. The long-term goal of this research project is to develop a rapid, sensitive, and specific multiplexed method to detect and quantify influenza virulence factors. The objective of the current proposal is to develop an oligonucleotide-based capture array to detect influenza RNA, thus allowing for virulence assessment, without amplification or labeling. This research will provide the scientific foundation for development of new screening methods that are critically needed for risk assessment in human influenza infections. The project will: 1)develop a hybridization strategy for virulence factors using synthetic oligonucleotides, 2) identify the virulent-associated PB1-F2 mutation in laboratory viruses, 3) identify the virulent- associated NA stalk motif in laboratory viruses, and 4) validate the SERS-based assay for influenza virulence factors in clinical samples. The innovative aspect of this proposal lies in the development of a novel nanofabrication method to produce a high sensitivity, rapid and reproducible platform technology for detection of influenza virulence factors, a capability that does not currently exist The proposal will take advantage of a well-defined SERS substrate platform, proven SERS sensing technology, and the integrated expertise of physical and biological investigators. At the conclusion of this project, it is our expectation that we will have developed and validated a nano-optical method for use as a powerful tool to detect influenza virulence factors. This project will ultimately demonstrate that bio-nanotechnology combined with vibrational spectroscopy has the ability to enhance detection and diagnosis of emerging viral infections with clinical applications. PUBLIC HEALTH RELEVANCE: There are several highly pathogenic influenza strains that are able to infect humans and are considered as potential precursors for future influenza pandemics. Unfortunately, to date there is no rapid and multiplexed method to screen for the biochemical markers of influenza virulence. The successful development of a rapid and sensitive method for screening of highly pathogenic influenza virulence factors would significantly improve our ability of identify emerging strains with a risk of high mortality, and allow public health professional to effectively respond to prevent widespread infection and/or death.